TISSUE ARRAY USING A CARRIER MEDIUM AND METHOD FOR PROVIDING THE SAME
An exemplary tissue array, and a method for producing the same, can be provided which can include providing an accepter biological structure(s), providing a donor tissue(s), removing a portion(s) of the donor tissue(s), and removing a portion(s) of the accepter biological structure(s). The removed portion(s) of the accepter biological structure(s) can have a size that is substantially similar to a size of the removed portion(s) of the donor tissue(s). The removed portion(s) of the donor tissue(s) can be inserted into the accepter biological structure(s) at a location substantially corresponding to the removed portion(s) of the accepter biological structure(s).
This application relates to, and claims priority from, U.S. Patent Application No. 62/034,235, filed on Aug. 7, 2014, the entire disclosure of which is incorporated herein by reference.
FIELD OF THE DISCLOSUREThe present disclosure relates generally to tissue arrays, and more specifically, to exemplary embodiments of a tissue array having tissue used for structural support.
BACKGROUND INFORMATIONMicroscopic analysis of tissue specimens can be the backbone of diagnostic surgical pathology, as well as being used in many research applications. Morphological analysis at the microscope can usually be performed by tissue sections being applied to microscopic slides. Tissue sections can be taken from tissue samples, which can be embedded in paraffin blocks. Paraffin embedding can be utilized in order to enable tissue sectioning for further analyses. Typically, paraffin blocks contain a single tissue specimen from a particular area, organ or tumor. Instead of having a single tissue or tumor embedded in a single paraffin block, it can be useful to have several different types of tissues embedded within the same block. This can ensure the proper choice of tissue, and can also reduce the number of sections to be taken, which would otherwise need to be taken from separate blocks.
Multi-tissue approaches can be important for analysis of new reagents, which need to be tested on several different types of tissue, as well as for the expression analysis of a particular antigen in various types of tissues or tumors. Paraffin blocks containing various tissues are often referred to as multi-tissue blocks or arrays, or as tissue microarrays (“TMAs”) when the size of the single tissue components is not bigger than a few millimeters. For a typical multi-tissue block, samples from different types of tumors are usually re-embedded in paraffin, and assembled into a new (e.g., multi-tissue) block. This can be done by taking already embedded paraffin tissue from blocks which serve as “donor” blocks, and by cutting out the to-be-sampled tumor/tissue areas with a scalpel or a dermatological circular punch device. The excised areas can be placed in a histological mold, and then re-embedded in paraffin. This can be done by taking the single paraffin tissue piece into a new embedding mold, which can then be heated up so that the paraffin melts and the tissues can be arranged in a way to ensure their proper orientation within the block. This can be important because a correct orientation, especially at the horizontal level, can be needed so that all tissue can be equally cut during the sectioning procedure.
However, a problem when creating a multi-tissue block can be providing a correct orientation of all the tissues and the correct placement in one level (e.g., an even surface). This can be important since all tissue pieces should be in one level so that the sectioning of the block can result in sections including all tissues and tissue pieces represented in a particular multi-tissue block.
Conventional multi tissue blocks generally consist of few tissues which are placed in a mold, heated to melt the paraffin and manually arranged into the desired position. The number and size of tissues in a multi-tissue block can be limited by the required minimum size of the assembled tissues. Additionally, the number of tissue samples can be limited because it can be more difficult to differentiate between different samples if there are too many samples in a small area that are not properly aligned and/or oriented. Further, the needed size of a multi-tissue block can be limited due to its use as a control tissue when placed next to other tissues onto the same slide. Multi-tissue blocks can be arranged in plain paraffin blocks as accepter media. By using circular punch devices, the size of the sample tissue can be reduced to small tissue cylinders. However, the diameter can be limited by the need to keep them in an upright position during the re-embedding process.
Though smaller tissue samples can increase the number of sampled tissues, the small size can make it difficult to keep them in an upright position, which can be needed for proper sectioning of all tissues in a multi-tissue block. The same problem can also be present in TMAs, where the tissue samples can usually be cylindrical punches of less than about 1mm in diameter. These punches can be placed in a plain paraffin block with punches of roughly the same size. The tissue punches have to remain in a vertical/upright position to ensure the proper presentation during sectioning of the block (e.g., during the removal of thin slices for examination). Due to the small diameter of the punches, and because they are placed in a paraffin block, the paraffin block with the tissue generally cannot be heated to ensure the proper embedding and polymerization of the paraffin. A plain paraffin microarray can only be slightly warmed such that the micro punches sticking out from the surface can be smoothly brought into one level by gently pushing them down and onto the level of the surrounding paraffin surface. If a plain paraffin microarray were heated to too high a temperature, the small-diameter tissue cores embedded therein cannot remain in a vertical position, and can become out of alignment in a horizontal position.
Prior methods of multi-tissue samples generally did not use a donor medium, except a paraffin-block only medium, which can be utilized for the embedding process. However, a plain paraffin block does not ensure the proper location and orientation of the tissues within a multi-tissue block. This can be done manually, and can be especially tedious and unsafe when the correct position of particular samples can be necessary.
Thus, it may be beneficial to provide an exemplary tissue arrangement, and method for making a tissue arrangement, that can easily maintain all of the tissue on the same level, and which can overcome at least some of the deficiencies described herein above.
SUMMARY OF EXEMPLARY EMBODIMENTSAn exemplary tissue array and a method for producing the same can be provided which can include, for example, providing an accepter block(s) having an accepter(s) tissue embedded therein, providing a donor block(s) having a donor tissue embedded therein, and removing a portion of the donor block(s) including a portion of the donor tissue(s). A portion of the accepter block(s) can be removed including a portion of the accepter tissue(s). The removed portion of the accepter block(s) can have a size that can be substantially similar to a size of the removed portion of the donor block(s). The removed portion of the donor block(s) can be inserted into the accepter block(s) at a location substantially corresponding to the removed portion of the accepter block(s).
In some exemplary embodiments of the present disclosure, the providing of the accepter block(s) can include inserting the accepter tissue into the accepter block(s), and heating the accepter block(s) to embed the accepter tissue into the accepter block(s). For example, the accepter biological structure(s) can be heated in a mold. The biological structure(s) can be pushed or compressed to the bottom of the mold in order to form a substantially flat surface thereon. The accepter biological structure(s) can be a single accepter biological structure and the removed portion(s) can include a plurality of removed portions. The donor tissue(s) can include a plurality of different donor tissues. In some exemplary embodiments of the present disclosure, the accepter biological structure(s) can include liver tissue, spleen tissue or any other tissue (e.g., any other homogeneous tissue).
The accepter block(s) can be heated after the removed portion of the donor block(s) can be inserted into the accepter block(s). The accepter block(s) can include paraffin. The removed portion of the donor block(s) and the removed portion of the accepter block(s) can be removed by a punch tool or a similar device, which can be shaped as a circle, a square, a rectangle, a triangle or any other shape
In another exemplary embodiment of the present disclosure, an exemplary tissue microarray can be provided, which can include an accepter block(s) which can include an accepter tissue and a donor tissue that can be substantially surrounded by, and substantially structurally supported by, the accepter tissue. In another exemplary embodiment of the present disclosure, an exemplary tissue array and a method for producing the same can be provided, in which accepter biological structure(s) and a donor tissue(s) can be provided, and portion(s) of the donor tissue(s) and portion(s) of the accepter biological structure(s) can be removed. The removed portion(s) of the accepter biological structure(s) can have a size that can be substantially similar to a size of the removed portion(s) of the donor tissue(s). The removed portion(s) of the donor tissue(s) can be inserted into the accepter biological structure(s) at a location substantially corresponding to the removed portion(s) of the accepter biological structure(s). The tissue arrangement can be a tissue microarray, which can include over 100 donor tissues. The accepter tissue(s) can include liver tissue, spleen tissue or any other tissue.
The biological structure(s) can be provided by inserting an accepter tissue(s) into the accepter biological structure(s), and heating the accepter biological structure(s) block(s) to embed the accepter tissue(s) into the accepter biological structure(s). The accepter biological structure(s) can be heated after the removed portion(s) of the donor tissue(s) can be inserted into the accepter biological structure(s). The accepter biological structure(s) can include paraffin. The removed portion(s) of the donor tissue(s) and the removed portion(s) of the accepter biological structure(s) can be removed by a punch tool.
In another exemplary embodiment of the present disclosure, an exemplary tissue arrangement can be provided which can include, for example, an accepter biological structure(s) that can include an accepter tissue(s) and a donor tissue(s). The donor tissue(s) can be substantially surrounded by, and substantially structurally supported by, the accepter tissue(s).
In another exemplary embodiment of the present disclosure, an exemplary tissue slide arrangement can be provided, which can include a slide(s) that can include a tissue arrangement(s). The tissue arrangement(s) can include an accepter tissue(s) and a donor tissue(s). The donor tissue can be substantially surrounded by, and substantially structurally supported by, the accepter tissue(s). The slide(s) can include a further tissue arrangement(s). The tissue arrangement(s) can be used as a control and can be compared to the further tissue arrangement(s). In certain exemplary embodiments of the present disclosure, the slide(s) can include a plurality of slides, where each of the slides can include a different reagent
In another exemplary embodiment of the present disclosure, an exemplary system and computer-accessible medium can be provided for creating a biological arrangement, in which, for example, an accepter biological structure(s) can be provided that can include an accepter tissue(s) surrounded by a support structure, a donor tissue(s) can be provided, and portion(s) of the donor tissue(s) can be removed. A boundary between the accepter tissue(s) and the support structure can be determined. Portion(s) of the accepter tissue(s) can be removed that can have a size that can be substantially similar to a size of the removed portion(s) of the donor tissue(s). The removed portion(s) of the donor tissue(s) can be inserted into the accepter tissue(s) at a location substantially corresponding to the removed portion(s) of the accepter tissue(s). The determining procedure can include differentiating between the accepter tissue(s) and the support structure such that only the accepter tissue(s) can be removed during the removal of the accepter tissue(s).
These and other objects, features and advantages of the exemplary embodiments of the present disclosure will become apparent upon reading the following detailed description of the exemplary embodiments of the present disclosure, when taken in conjunction with the appended claims.
Further objects, features and advantages of the present disclosure will become apparent from the following detailed description taken in conjunction with the accompanying Figures showing illustrative embodiments of the present disclosure, in which:
Throughout the drawings, the same reference numerals and characters, unless otherwise stated, are used to denote like features, elements, components or portions of the illustrated embodiments. Moreover, while the present disclosure will now be described in detail with reference to the figures, it is done so in connection with the illustrative embodiments and is not limited by the particular embodiments illustrated in the figures or the appended claims.
DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTSExemplary method and apparatus, according to an exemplary embodiment of the present disclosure, can provide and/or ensure that the orientation and proper placement of tissues for use, for example, in a tissue array or a TMA, can be secured when assembled into a multi tissue block. The exemplary method and apparatus can utilize various pieces or portions of accepter tissue (e.g., large or other sized pieces of tissue) that can be placed in a cassette. The exemplary accepter tissue can then be processed similar to normal tissue undergoing a standard paraffin embedding process resulting in a standard paraffin block. Alternatively, the accepter tissue and the donor tissue can be frozen in an ice block. Alternatively, the accepter tissue can be any other suitable medium which can serve as a support for the donor tissue.
As shown in
The exemplary accepter block 100 can then serve as an accepter for the other samples taken from one or more donor blocks. For example, as shown in
The exemplary tissue array can be created or otherwise provided using the following exemplary method. For example, tissue 205 from donor block 200 can be removed from a desired area and with a desired size. For example, a portion 215 of donor tissue 205 can be removed through a hole 210 created in donor block 200. This can be performed, for example, with various dermatological punch devices, which can be, for example, circular knives in various diameters. Alternatively, exemplary punch devices shaped other than circular can be used (e.g., square, rectangle, triangle etc.). Punch devices of the same or similar size/diameter/shape can then be used to extract a similarly sized circular area 115 from a hole 110 in accepter block 100. The punched out area 115 from accepter block 100 can be discarded or used for other applications. The empty area within accepter block 100 can then be filled with the portion of tissue 215 of a similar size and shape taken from donor block 200. This procedure can be repeated with various different donor samples, and can be done with variably-sized donor tissue.
When all donor samples are excised and placed into similar sized empty spaces in the accepter tissue, an accepter block with several circular samples is created. In an exemplary embodiment of the present disclosure, the exemplary donor punches can fit and match neatly in the pre-punched spaces of the accepter block. The accepter block formed from paraffin wax, accepter tissue, and donor tissue (e.g., accepter block 300 of
In conventional tissue arrays, the tissue may not be easily maintained in one level, if at all. This can result in, for example, all of the various tissues sticking out of the block, and the tops of the various tissues being at different levels. Thus, to obtain a tissue sample containing all the tissues embedded in a prior art tissue block, various levels have to be removed until a block is achieved having all of the tissues on the same level. This can involve iteratively cutting and discarding the top layer until the desired top layer is achieved having all of the tissues on the same top level. Thus, conventional blocks can be very wasteful as a significant portion of the tissue has to be removed to achieve a block having all of the tissue on the same level. In contrast, the exemplary method and apparatus can easily put or maintain the tissues in one level during the exemplary re-embedding process, and little to no tissue is wasted. Prior art methods attempted to place the tissue on one level during the re-embedding process. However, such attempts were generally unsuccessful, and any attempted improvement in the maintaining of the tissues on the same level was tedious and time consuming.
The exemplary method and apparatus, according to an exemplary embodiment of the present disclosure, can also be used as an exemplary test or control tissue (e.g., a positive control or a negative control). For example, when testing and/or staining the tissue of a subject or person, it can be beneficial to ensure (a) that the correct reagent was used; (b) that the correct amount of reagent was used (e.g., no overstaining or understaining); and (c) that the reagent had not expired or lost is effectiveness. Without a control tissue, a person cannot tell if the reagent used is still viable, or if the correct amount of reagent was used. As the exemplary method and apparatus can place more tissue samples in a smaller space (e.g., 2, 3, 4, 5, 6, 7, etc. tissue samples in a small confined space), more tissue samples can be placed on a slide as a control, as less space can be needed. In contrast, conventional tissue arrays need a larger area for the same amount of tissue samples, and thus may not be suitable for use as control tissues.
The exemplary method and apparatus, according to an exemplary embodiment of the present disclosure, can utilize a mechanical device (e.g., manual or automated), which can sample punches of very small diameters (e.g., about 0.3 mm) to larger diameter punches (e.g., about 1 mm), although smaller and larger punch sizes can be utilized. These exemplary micro-punches can be taken from the donor tissues and placed into to an accepter paraffin block. Because of the size of the tissue donor punches, several hundred of array punches can be placed into an accepter block. As an accepter, a plain paraffin block can be used. As described herein, the exemplary method and apparatus can utilize the accepter block which can contain an already embedded piece of donor tissue. Instead of using a plain paraffin block without any tissue inside (e.g., prior art accepter blocks), the exemplary method and apparatus can utilize accepter tissue, which can serve as a scaffolding for the micro-tissue punches to surround and support the donor tissue. The accepter tissue can also act as a test tissue, and can include tissue obtained from the liver, spleen or similar homogeneous tissues, although other tissue types can be used. Additionally, mediums other than tissue can be used as an accepter medium including biological and non-biological mediums (e.g., gels containing organic matter such as collagen, and/or a mixture of organic matter and artificial material such as polymers and/or plastics).
As shown in
While conventional tissue blocks can only be slightly warmed, the exemplary tissue block can be heated until the paraffin is completely melted, and the accepter tissue containing the micro-punches can be brought in one level, which can be, for example, the bottom of the mold. This can ensure the proper placement of all punches in the level of the tissue. Furthermore, for example, for appropriate and smooth cutting, the complete melting and solidifying of the paraffin can be beneficial. With the conventional method, cutting of tissue arrays can often be tedious and difficult. This can be because the small paraffin cylinders are placed into paraffin, and are only held in place by physical forces. This can cause problems during the sectioning process, as single punches can shift or be lost during the sectioning process. If the conventional plain paraffin tissue array block is heated, the donor micro punches (e.g., several hundred) can be in disarray and can tip over. By using an accepter tissue that can structurally hold the inserted micro-punches in place, the exemplary tissue array, according to an exemplary embodiment of the present disclosure, can be treated like a standard paraffin block without tissue, and can be heated so that the paraffin can be melted. Thus, fully melted paraffin can then be brought down in temperature, and can solidify to ensure the homogenous polymerization of all the paraffin within the exemplary tissue array block. This can make the sectioning process easier. Consequently, a higher yield of tissue sections can be gained by the exemplary method.
The exemplary method can also be used to produce one or more TMAs. Generally, the TMA can include hundreds of small tissue samples (e.g., 200-300 samples, although not limited thereto). Some conventional TMAs (e.g., TMAs 3205, 3305 of
In contrast to conventional TMAs (e.g., TMAs 3205, 3305), the exemplary TMA 2805 (see, e.g.,
The sections of the exemplary tissue array can also be placed in a water bath without affecting the tissue samples embedded therein. For example, when a tissue array is sliced, the slice can be placed in a water bath. Tissue samples in the conventional tissue array can easily move or shift during this process. Once applied to a glass slide for histological analysis, the tissues can be or become missing and/or misaligned and deranged. (See e.g.,
An additional problem with the conventional TMAs can be that the inserted donor tissues can have different thicknesses. The use of the TMA can be limited by the thinnest inserted tissue. Once this tissue, or several tissues, with less thickness than the other tissues have reached the section level, the TMA can lack those tissues.
In contrast to the conventional TMAs, in the exemplary TMA, the tissue samples from a donor tissue, having only limited thickness left, can be punched several times, and stacked into a single punch hole of the accepter tissue of the exemplary TMA Thus, facilitating the use of thin donor tissues as several punches from the same thin donor tissue, which can now be placed on top of each other in a new TMA, rendering it sufficiently thick and creating a new TMA with a homogeneous thickness of all represented tissue. Therefore, the exemplary TMA, according to an exemplary embodiment of the present disclosure, can include several layers of donor tissue that can be placed on top of each other to increase the thickness of the donor tissue in the accepter block. This can facilitate a similar thickness of all donor tissue specimens. This can also increase the usability of the exemplary donor block since many more sections can be taken from the donor block while still displaying all the inserted donor tissues.
While the conventional tissue arrays and TMAs are manually arranged, the exemplary tissue array, according to the exemplary embodiments of the present disclosure, can be provided using, for example, an automated process, as the accepter tissue can provide a reference for the placement of the donor tissue. For example, after the accepter block has been created (e.g., using the exemplary procedure described above using a manual or automated process), the boundary of the accepter tissue and the donor tissue can be automatically determined, using, for example, an exemplary boundary determination procedure according to an exemplary embodiments of the present disclosure. When the boundary has been determined for the exemplary accepter tissue and the exemplary donor tissue, holes can be punched into to the accepter tissue using an exemplary hole punching procedure. Since an automated procedure can be more accurate, more samples can be placed in an accepter tissue through the exemplary automated procedure.
After the holes have been punched in the accepter tissue using the exemplary automated procedure of an exemplary embodiment of the present disclosure, tissue samples can be removed from one or more donor tissues and placed into the accepter tissue. The exemplary tissue array can then be heated to place all of the tissue samples on a single level (e.g., by compressing the tissue). Thus, while the exemplary tissue arrays can be created and/or provided using, for example, a manual procedure, the exemplary tissue arrays can also be created using an automated procedure, facilitating a quicker and cheaper process for creating tissue arrays.
As shown in
Further, the exemplary processing arrangement 3602 can be provided with or include an input/output arrangement 3614, which can include, for example a wired network, a wireless network, the internet, an intranet, a data collection probe, a sensor, etc. As shown in
The foregoing merely illustrates the principles of the disclosure. Various modifications and alterations to the described embodiments will be apparent to those skilled in the art in view of the teachings herein. It will thus be appreciated that those skilled in the art will be able to devise numerous systems, arrangements, and procedures which, although not explicitly shown or described herein, embody the principles of the disclosure and can be thus within the spirit and scope of the disclosure. Various different exemplary embodiments can be used together with one another, as well as interchangeably therewith, as should be understood by those having ordinary skill in the art. In addition, certain terms used in the present disclosure, including the specification, drawings and claims thereof, can be used synonymously in certain instances, including, but not limited to, e.g., data and information. It should be understood that, while these words, and/or other words that can be synonymous to one another, can be used synonymously herein, that there can be instances when such words can be intended to not be used synonymously. Further, to the extent that the prior art knowledge has not been explicitly incorporated by reference herein above, it is explicitly incorporated herein in its entirety. All publications referenced are incorporated herein by reference in their entireties.
Claims
1. A method for providing a biological arrangement, comprising:
- providing at least one accepter biological structure;
- providing at least one donor tissue;
- removing at least one portion of the at least one donor tissue;
- removing at least one portion of the at least one accepter biological structure, wherein the at least one removed portion of the at least one accepter biological structure has a size that is substantially similar to a size of the at least one removed portion of the at least one donor tissue; and
- inserting the at least one removed portion of the at least one donor tissue into the at least one accepter biological structure at a location substantially corresponding to the at least one removed portion of the at least one accepter biological structure.
2. The method of claim 1, wherein the at least one biological structure can be provided by inserting at least one accepter tissue into the at least one accepter biological structure, and heating the at least one accepter biological structure to embed the at least one accepter tissue into the at least one accepter biological structure.
3. The method of claim 2, further comprising heating the at least one accepter biological structure in a cassette.
4. The method of claim 2, further comprising at least one of pushing or compressing the at least one accepter biological structure so as to form a substantially flat surface thereon.
5. The method of claim 1, wherein the at least one accepter biological structure is a single accepter biological, and the at least one removed portion includes a plurality of removed portions.
6. The method of claim 5, wherein the at least one donor tissue includes a plurality of different donor tissues.
7. The method of claim 1, wherein the at least one accepter biological structure includes at least one of (i) liver tissue, (ii) spleen tissue, or (iii) a homogeneous tissue.
8. The method of claim 1, further comprising heating the at least one accepter biological structure after the at least one removed portion of the at least one donor tissue is inserted into the at least one accepter biological structure.
9. The method of claim 1, wherein the at least one accepter biological structure includes paraffin.
10. The method of claim 1, wherein the at least one removed portion of the at least one donor tissue and the at least one removed portion of the at least one accepter biological structure is removed by a punch tool.
11. The method of claim 10, wherein the punch tool is shaped as at least one of (i) a circle, (ii) a square, (iii) a rectangle, or (iv) a triangle.
12. A tissue arrangement, comprising:
- at least one accepter biological structure which includes at least one accepter tissue and at least one donor tissue, wherein the at least one donor tissue is substantially surrounded by, and substantially structurally supported by, the at least one accepter tissue.
13. The tissue arrangement of claim 12, wherein the tissue arrangement is a tissue microarray.
14. The tissue arrangement of claim 13, wherein the tissue microarray includes over 100 donor tissues.
15. The tissue arrangement of claim 12, wherein the at least one accepter tissue includes at least one of (i) liver tissue, (ii) spleen tissue, or (iii) a tissue.
16. A tissue slide arrangement, comprising:
- at least one slide which includes at least one tissue arrangement, the at least one tissue arrangement including at least one accepter tissue and at least one donor tissue, wherein the at least one donor tissue is substantially surrounded by, and substantially structurally supported by, the at least one accepter tissue.
17. The tissue slide arrangement of claim 16, wherein the at least one slide includes at least one further tissue arrangement, and wherein the at least one tissue arrangement is used as a control to be compared to the at least one further tissue arrangement.
18. The tissue slide arrangement of claim 16, wherein the at least one slide includes a plurality of slides, and each of the slides includes a different reagent.
19. A non-transitory computer-accessible medium having stored thereon computer-executable instructions for creating a biological arrangement, wherein, when a computer arrangement executes the instructions, the computer arrangement, upon an execution of the instructions, is configured to perform procedures comprising:
- providing at least one accepter biological structure including at least one accepter tissue surrounded by a support structure;
- providing at least one donor tissue;
- removing at least one portion of the at least one donor tissue;
- determining a boundary between the at least one accepter tissue and the support structure;
- removing at least one portion of the at least one accepter tissue, wherein the at least one removed portion of the at least one accepter tissue has a size that is substantially similar to a size of the at least one removed portion of the at least one donor tissue; and
- inserting the at least one removed portion of the at least one donor tissue into the at least one accepter tissue at a location substantially corresponding to the at least one removed portion of the at least one accepter tissue.
20. The computer-accessible medium of claim 19, wherein the determining procedure includes differentiating between the at least one accepter tissue and the support structure such that only the at least one accepter tissue is removed during the removal of the at least accepter tissue.
Type: Application
Filed: Aug 7, 2015
Publication Date: Aug 17, 2017
Patent Grant number: 10948390
Inventors: Achim JUNGBLUTH (New York, NY), DENISE FROSINA (Bay Terrace, NY)
Application Number: 15/502,059